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Deletion of RBP-J within renin cells of Bl6 and SV mice leads to B-cell leukemia and MPD, respectively. (A) Representative flow cytometry plots performed on the bone marrow of control and mutant mice from the SV (left panel) and Bl6 (right panel) background. Conditional deletion of RBP-J within renin cells of SV mice results in decreased number of CD19+B220+ B cells and an increase in CD11b+Gr1− and CD11b+Gr1+ myeloid cells. Conversely, conditional deletion of RBP-J within renin cells of Bl6 mice results in an aberrant population of CD19+B220dim leukemic B cells and a decrease in myeloid cells. (B) Mutant animals from the Bl6 background have increased spleen weight, liver weight and white blood cell count, as well as decreased hemoglobin, compared with mutant animals from the SV background. Further, mutant SV animals with two copies of Cre recombinase have increased spleen weight, increased white blood cell count and decreased hemoglobin compared with mutant SV animals with one copy of Cre recombinase. There was a trend towards worse disease in mutant Bl6 animals with two copies of Cre recombinase compared with Bl6 animals with one copy of Cre recombinase, although this was not statistically significant. *P<0.05, **P<0.01 and ***P<0.001, Mann–Whitney U-test. The sample size for each group is shown. (C) Animals with two copies of Cre recombinase (RBP-Jfl/fl;Ren1dCre/Cre) have a higher incidence of B-cell leukemia compared with mice with only one copy of Cre recombinase (RBP-Jfl/fl;Ren1dCre/+) for both the SV and Bl6 background. Mutant animals from the Bl6 background predominantly develop B-cell leukemia, whereas mutant animals from the SV background have a higher incidence of MPD (P<0.001, Fisher's exact test). The sample size for each group is shown. (D) In both SV and Bl6 mice, survival decreases in mutant animals with two copies of Cre recombinase (RBP-Jfl/fl;Ren1dCre/Cre) compared with those with only one copy (RBP-Jfl/fl;Ren1dCre/+). The sample size for each group is shown.

Deletion of RBP-J within renin cells of SV mice leads to dermatitis. (A-C) Mutant animals from the SV background develop hair loss over their back (A) and cystic nodules on their snouts (B) and tails (C). (D-F) With age, hair loss (D) progresses to ulceration and hyperkeratinization (E,F). Pictures are of the same mous­e at day of life 57 (D), day of life 107 (E) and day of life 155 (F). (G-I) Hematoxylin and Eosin staining demonstrates thickened epidermis and dermis, as well as large cysts and inflammatory infiltrates, in mutant animals (H,I) compared with control animals (G). Scale bars: 200 µm.

Dermatitis in SV mice is due to deletion of RBP-J within progenitor cells of the skin and the MPD is a secondary, reactive process. (A) Renin lineage (GFP+) cells are present in the skin of RBP-Jfl/+;Ren1dCre/+(SV);mTmG control mice (left). The nodular and cystic skin lesions of RBP-Jfl/fl;Ren1dCre/+(SV);mTmG mutant mice are partially GFP positive, signifying the presence of Cre recombinase and thus deletion of RBP-J within skin lesion cells (right). Scale bars: 200 μm. (B) PCR was performed on genomic DNA from bone marrow cells from control animals, animals with B-cell leukemia and animals with dermatitis/MPD, using primers designed to detect wild-type RBP-J alleles, floxed RBP-J alleles and RBP-J alleles that have undergone Cre-mediated deletion. The bone marrow cells from animals with MPD show predominantly floxed RBP-J alleles, but not deletion alleles, confirming that the myeloid cells have not undergone deletion of RBP-J. (C) Flow cytometry on bone marrow cells from mutant Bl6 animals shows a large population of CD19+B220dim leukemia cells, which are largely GFP positive (left panel). Conversely, the bone marrow from mutant SV mice has an increased population of CD11b+Gr1+ myeloid cells, which are RFP positive (right panel), and thus have not undergone Cre-mediated recombination of RBP-J.

Bl6 mice have more renin lineage cells within hematopoietic tissues compared with SV mice. (A) Bl6 mice have a trend towards more renin lineage cells (marked by GFP) in the spleen compared with SV mice (P=0.057, Mann–Whitney U-test). There is a non-significant trend towards more renin lineage cells in the bone marrow as well (P=0.076, Mann–Whitney U-test). The sample size for each group is shown. (B) There are more renin lineage cells (GFP+) in the peripheral blood of Bl6 mice compared with SV mice throughout life. (C) Immunofluorescent images of renin lineage (GFP+) cells in the spleen and liver of newborn SV mice compared with newborn Bl6 mice. Scale bars: 100 µm.

Renin-expressing cells are a susceptible population for malignant transformation. (A) Comparison of the expression pattern of different B-cell Cre recombinase transgenes – Mb1-Cre, CD19-Cre and renin-Cre – within the bone marrow and spleen by flow cytometry. Within the bone marrow and spleen, Mb1Cre;mTmG mice have the highest number of B cells that express GFP, followed by CD19Cre;mTmG and Ren1dCre;mTmG. *P<0.05 and **P<0.01, Kruskal–Wallis test. The sample size for each group is shown. (B) Cre-mediated GFP expression in the spleens of Mb1Cre;mTmG, CD19Cre;mTmG and Ren1dCre;mTmG mice. Scale bars: 200 µm. (C) Conditional deletion of RBP-J within renin-expressing cells leads to the development of B-cell leukemia and early death. However, conditional deletion of RBP-J within Mb1-expressing or CD19-expressing cells does not lead to B-cell leukemia. At advanced ages, 20% of RBP-Jdel/fl;Mb1Cre/+ mice and 50% of RBP-Jdel/fl;CD19Cre/+ mice developed abdominal tumors, leading to abdominal distension and death. The sample size for each group is shown. (D) Conditional deletion of RBP-J within renin-expressing cells, but not Mb1-expressing or CD19-expressing cells, resulted in elevated spleen weight, liver weight and white blood cell count and decreased hemoglobin compared with littermate control mice. *P<0.05 and **P<0.01, Mann–Whitney U-test. The sample size for each group is shown. (E) Representative tumor from RBP-Jdel/fl;CD19Cre/+ mice (leftmost). By flow cytometry analysis, abdominal tumors were positive for CD5 but negative for B-cell and myeloid markers. Further, the tumors were RFP+ and thus not due directly to deletion of RBP-J. FSC, forward scatter; SSC, side scatter.

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